The transmissible spongiform encephalopathies (TSEs) are characterized by the pathogenic accumulation of the protease-resistant form of prion protein (PrP-res). To define how PrP-res is made, we have extended our studies with a cell-free conversion reaction in which PrP-res promotes the conversion of the normal protease-sensitive prion protein (PrP-sen) to PrP-res. Recent refinements in this conversion reaction have allowed us to 1) greatly improve the efficiency of the conversion, 2) perform the conversion under nondenaturing conditions, and 3) induce the conversion under conditions more like those in vivo, i.e., using scrapie-infected, slide-mounted brain slices or crude brain homogenates. Denaturation experiments have identified a particularly stable 16-kDa domain of PrP-res that appears critical to the converting activity of PrP-res. To further characterize the species specificity of the conversion reaction, PrP-res derived from BSE-infected cattle and scrapie-infected sheep were reacted with PrP-sen from a variety of species. BSE PrP- res converted human PrP-sen to PrP-res, but 10-fold less efficiently than it converted the homologous bovine PrP-sen. This result suggests that if dietary BSE PrP-res is able to get to the brains of humans, there is a finite potential that pathogenic PrP-res formation could be initiated. Sheep PrP-res readily converted both sheep and bovine PrP-sen to PrP-res. Interestingly, a comparison of the conversions of two different sheep PrP-sen genotypes by sheep PrP-res showed that the genotype of PrP-sen associated with scrapie-susceptibility was more efficiently converted than the resistant genotype. Using scrapie-infected mouse neuroblastoma cell cultures, we have identified several new glycosaminoglycan (GAG)-analog inhibitors of PrP-res formation. Several of these represent a new class of inhibitor that offers several possible therapeutic advantages over previously known inhibitors. Large differences in GAG content were observed between scrapie-infected and control cells, providing more clues that GAGs are important in TSEs.